We study the mechanism and regulation of protein synthesis in eukaryotic cells. Of special interest are the regulation of protein synthesis by GTP-binding (G) proteins and protein phosphorylation. In addition, we are studying unusual post-translational modifications of the factors that assist the ribosome in synthesizing proteins. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is composed of three subunits including the G protein eIF2gamma. During translation initiation, the GTP bound to eIF2gamma is hydrolyzed to GDP, and the factor eIF2B recycles eIF2-GDP to eIF2-GTP. Phosphorylation of eIF2alpha on serine 51 coverts eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2 (activated by amino acid starvation), PKR (activated by double-stranded RNA in virally infected cells), PERK (activated by ER stress), and HRI (activated under conditions of low heme). Our structure-function studies on eIF2 have provided insights into human disease. Protein synthesis plays a critical role in learning and memory in model systems, and our studies have linked a human X-linked intellectual disability (XLID) syndrome to altered function of eIF2. In previous studies with collaborators in Israel and Germany, we characterized a human XLID syndrome characterized by intellectual disability and microcephaly. The patients carry a mutation in the EIF2S3 gene encoding eIF2gamma, and genetic and biochemical studies revealed that the mutation disrupts eIF2 complex integrity and translation start codon selection. More recently, working with collaborators in Germany, Slovakia, and at Walter Reed National Military Medical Center, we characterized two additional mutations in eIF2gamma found in patients exhibiting intellectual disability, epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity. Based on this constellation of phenotypes, the disease has been termed MEHMO syndrome, and we now conclude that MEHMO syndrome is caused by mutations in EIF2S3. Over the past year we have generated a yeast model of a recently identified mutation in eIF2gamma that causes MEHMO syndrome. The mutation maps to the tRNA binding site in eIF2gamma and genetic and biochemical studies revealed impaired eIF2 function, altered translational control of specific mRNAs, and reduced stringency of translation start site selection. Based on our studies we propose mutations in eIF2gamma impair the efficiency and fidelity of protein synthesis, and that this altered control of protein synthesis underlies MEHMO syndrome. Our second major research focus involves the translation factor eIF5A,the sole cellular protein containing the unusual amino acid hypusine. Using molecular genetic and biochemical studies, we showed that eIF5A promotes translation elongation, and that this activity is dependent on the hypusine modification. We also showed that eIF5A from yeast, like its bacterial ortholog EF-P, stimulates the synthesis of proteins containing runs of consecutive proline residues. Consistent with these in vivo findings, we showed that eIF5A was critical for the synthesis of polyproline peptides in reconstituted yeast in vitro translation assays. Using misacylated tRNAs we showed that proline and not tRNA(Pro) imposes the eIF5A requirement; and we also showed that the more flexible proline analog azetidine-2-carboxylic acid relaxes the eIF5A requirement for peptide synthesis. Using directed hydroxyl radical probing experiments, we mapped eIF5A binding near the E site of the ribosome, and then working with x-ray crystallographers in France, we obtained the crystal structure of eIF5A bound to the yeast 80S ribosome. The structure revealed interactions between eIF5A and conserved ribosomal proteins and rRNA bases. Moreover, eIF5A occupies the E site with the hypusine residue projecting toward the acceptor stem of the P-site tRNA. In related studies, we reported the structure of a diproline-tRNA analog bound to the ribosome, revealing that proline affects nascent peptide positioning in the ribosome exit tunnel. Taken together, our studies support a model in which eIF5A and its hypusine residue function to reposition the acceptor arm of polyprolyl-tRNA in the P site to alleviate stalling and that the body of eIF5A functions like polyamines to enhance general protein synthesis. In collaboration with researchers at Johns Hopkins University, we reported that in addition to its critical requirement for polyproline synthesis eIF5A functions globally to promote both translation elongation and termination. Over the last year, we have linked eIF5A to the regulation of polyamine metabolism in mammalian cells. The enzyme ornithine decarboxylase (ODC) catalyzes the first step in polyamine synthesis. ODC is regulated by a protein called antizyme, which, in turn, is regulated by another protein called antizyme inhibitor (AZIN1). The synthesis of AZIN1 is inhibited by polyamines and this regulation is dependent on a conserved element in the 5 leader of the AZIN1 mRNA that we refer to as a uCC - for upstream conserved coding region. Whereas translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate start codons, we found that high polyamines enhance translation initiation from the near-cognate start site of the uCC. Surprisingly, this regulation is dependent on the sequence of encoded polypeptide. Ribosome profiling revealed polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in the uCC. Mutation of the PPW motif impaired initiation at the near-cognate AUU start codon of the uCC and abolished polyamine control, leading to constitutive high-level expression of AZIN1. In contrast, substituting an alternate elongation pause sequence restored uCC translation. We proposed that most scanning ribosomes bypass the near-cognate start codon of the uCC without initiating and then translate AZIN1. However, occasionally a ribosome will initiate translation at the uCC start codon. Under conditions of high polyamines, these elongating ribosomes pause on the PPW motif. This paused ribosome serves as a roadblock to the scanning ribosomes that bypass the near-cognate start codon. The resultant queuing of scanning ribosomes behind the paused elongating ribosome positions a ribosome near the start site of the uCC providing greater opportunity for initiation at the weak start site. Consistent with the notion that ribosome queuing is important for uCC translation, impairing ribosome loading reduced uCC translation and derepressed AZIN1 synthesis. We believe that this mechanism of a paused elongating ribosome promoting initiation at an upstream weak start site via ribosome queuing may underlie the control of translation of other mRNAs, especially those whose translation is derepressed by conditions that impair ribosome loading. In further studies on the AZIN1 regulatory mechanism, we identified eIF5A as a sensor and effector for polyamine control of uCC translation. Using our reconstituted in vitro translation assay system, we found that synthesis of a PPW peptide, like translation of polyproline sequences, requires eIF5A. Moreover, this ability of eIF5A to stimulate PPW synthesis was inhibited by polyamines. Thus, we propose that polyamine inhibition of eIF5A serves as the trigger to cause the ribosome pause that governs uCC translation. Taken together, our studies have shown that eIF5A functions generally in protein synthesis and that modulation of eIF5A function by polyamines can be exploited to regulate specific mRNA translation.